BR112015020697A2 - novel bacteriophage and antibacterial composition comprising the same - Google Patents

Abstract

"novel bacteriophage and antibacterial composition comprising the same" The present invention relates to a novel bacteriophage 1cj22 (kccm 1 1364p). The present invention further relates to an antibacterial composition comprising bacteriophage dcj22 (kccm1 1364p) as an active ingredient. furthermore, the present invention provides a method for preventing and / or treating infectious diseases caused by closiridium perfrngens in animals other than humans using the bacteriophage ddcj22 (kccm 11364p) or the antihacteric composition comprising the bacteriophage dcj22 (kccm 1 1364p ) as an active ingredient

Description

‘'NEW BACTERIOPHAGUS AND ANTIBACTERIAL COMPOSITION UNDERSTANDING THE SAME'’

Technical Field

The present invention relates to a new bacteriophage with a specific bactericidal activity against the pathogen Cáwvásm pebrfegtw, and an antibacterial composition comprising the same. The present invention also relates to a method of preventing and treating animal diseases using the new baeteriophane or antibacterial composition.

Background to the Invention

Cfevnafem (CP), which is an obligatory large granipositive anaerobic bacillus, is known as a bacterium that does not have flagellum and forms a spore. Q CfoAndws /w#wgcn.v. which is a bacteria that causes diarrhea, or the like, particularly in domestic animals such as chicken, pork, etc., and the like, has been recognized as one of the dangerous and fatal pathogenic bacteria in the animal industry, such as causing typhoid fever avian.

Currently, one of the diseases frequently generated in the poultry and pork industries is the entangling necrotic by Goxmàfíw? It is known that necrotic enteritis is often generated by a -infection of Chs / rhyme perfmgtw and Coeeúfe », and as the main symptom of the necrotic entente, it produces bloody diarrhea due to serious necrodic lesions in a lower portion of the small intestine of chickens , pigs and the like.

Ecr-necrotic entente generates symptoms of dehydration, periodic diarrhea and similar symptoms in the infected animal, according to the severity of the disease, and gradually weakens the animal's body, causing growth retardation, among other problems, in such a way that the Necrotic enteritis has become a significant problem in the animal industry. Also, 25 like Cfo.wrá: / fetf? / Moringen.? It is easily propagated through animal feces,

2/28 animal-to-animal transmission in a common breeding space can be easily generated by oral infection through soil, contaminated food or similar situations. In particular, the incidence of young ran animals is high, so that perfection has become a major problem.

In turn, the bacteriophage is a specialized type of virus that infects and destroys only bacteria, and can self-replicate only within host bacteria. The bacieríófago has strong host specificity in comparison with antibiotics and, recently, the appearance of an antibiotic resistant strain has become a serious problem, so that the interest in the practical use of the hacteriophage has increased ΙΌ (Non-patent documents I and 2).

Therefore, research on bacteriophages has been actively conducted in several countries around the world and in addition to patent applications for bacteriophages. there was a gradual increase in approval requests sent to the Food and Drug Administration (FDA) for compositions containing the baeteriotago.

With regard to bacteriophages available in the technical state, the Patent Document reveals a baeteriotago .with a specific baciericide activity against C / osa-àAmí? perpetual. and Patent Document 2 discloses a baeteriophagus with specific bactericidal activity against asthma. In addition to these, Patent Document 3 describes a lyrical protein derived from a baeteriotago that specifically destroys the peptidogiicano structure of the bacterial cell membrane, and bacteria Used by the lithium protein.

E-ntrentanto, despite the existence of the aforementioned documents in the state of the art, a technology associated with the baeteriotago to prevent and / or treat infectious diseases, particularly the necrotic entity by Clostridium jxfrfrmg & w, which is still an important problem 25 in the animal industry, including the poultry and pork industries, is still insufficient.

3/28 so that a bacteriophage and a technology associated with the bacteriophage - must be developed.

Documents available in technical status

Dock meo tos Pa tea ta rios

- Patent Document 1: Korean patent published No. 10-2'01.2-0076710 A

-Patentary Document 2: Korean registered patent No. 10-0910961 BI

-Patentary Document 3: Korean patent published No. 10-2009-0021475 A

Patent Documents

-Document No Patent I: C / sZoM e / arA, Arch. Z / me »/. 77? Er. Ζ2φ. 2.275- / ¾ /.9R7

- Non-Patent Document 2; Sm ·? # /-/Oo.IGH-? Áríw et oZ, «owí <wfo7órímç a / temorívost ÃioíEave / <> /. 7 0625, 2005, 2JZZ.Z0 Disclosure

Technical problem

The inventors of the present invention have carried out studies to solve problems such as the presence of antibiotic resistant bacteria, e.g. presence of antibiotics remaining in the meat, in addition to effectively preventing and treating infectious diseases caused by Cfostríc / àrm and, as a result, the inventors of the present invention isolated the new bacteriophage ΦΟ22 (KCCMl 1364-P) which has a specific bactericidal activity against tVvbrídmm perÁb? gén.y present- in nature.

2'0 In addition, the inventors of the present invention have identified morphological, biochemical and. genetics of the new bacteriophage and confirmed that the bacteriophage has excellent resistance to acids, heat resistance, resistance to drought and similar conditions, thus developing an antibiotic, a disinfectant, - a food additive, and other compositions using the new bacteriophage. In addition, the inventors of the present invention have developed a composition for the prevention or treatment of

4/28 Cfosffitâwn / wynugmv infectious diseases and a method to prevent or treat the disease using the composition.

The present invention aims to provide a new bacteriophage ΦΟ22 (KCCM1I364P) with a bactericidal activity. Specific against C / tw / rôfe »? Pe / coldgens.

The present invention further aims to provide a composition for the prevention and / or treatment of infectious diseases by Cfosirtâiiím perpagageas containing the bacteriophage ΦΟΙ22 (KCCM1I364P) as an active ingredient.

In addition, the present invention aims to provide an antibiotic, a food additive, a water additive, potable · a disinfectant or a cleaning product containing bacteriophage ΦΟ22 (KCCM113641 · *) as an active ingredient.

In addition, the present invention aims to provide a method of prevention and / or treatment of infectious diseases by Cfatfrtâfam / wyHwgw in animals, except for humans, using the bacteriophage ΦΟ22 (KCCM11364?) Or a bacteriophage-containing composition ΦΟ22 (KCCM II364?) As an active ingredient.

Technical Solution

According to one embodiment, the present invention provides a new bacteriophage <Í * CJ22 (KCCM 11364?) With a specific baetericidal activity against

According to another embodiment, the present invention provides a composition .20 for the prevention or treatment of infectious disease caused by Cfemrk & Km, said composition containing bacteriophage Φ <?, 122 (Kt t M11364P) as an active ingredient, as described above.

According to another configuration, the present invention provides an antibiotic, a food additive, an additive for drinking water, a disinfectant or a cleaning product containing the bacteriophage Φ € Ι2.2 (KCCM 11364?) Containing an active ingredient,

5/28 as described above

According to another embodiment, the present invention provides a method of preventing or treating infectious disease caused by CZmrrüÃM / w jjer / nwgem comprising administration of bacteriophage <PCJ22 (KCCMlI36-4P) or of composition 5 containing bacteriophage $ CJ22, as described above, for animals, except for humans.

Advantageous Effects

The bacteriophage $ CJ22 (KCCMI136-4P) according to the present invention can have a specific bactericidal effect against Cfes / mZ? W perfetogrw.

In addition, the bacteriophage <bCJ22 (K.CCMH364P) according to the present invention has excellent acid resistance, heat resistance and resistance to drought, such that the bacteriophage <hC.I22 (KCCM i 13640) can be used to prevent or treat infectious diseases by CtotoWíwí at various levels of temperature or pH and humidity conditions, and can be used as an antibiotic, a food additive.

tmi additive for drinking water, a disinfectant, a similar cleaning product.

Furthermore, according to the present invention, infectious diseases caused by Ctostoàfnw can be prevented or treated by administering the bacteriophage <! * C.I22 (KCCM 11364P) or the composition containing the bacteriophage <1022 (KCCMI1364I ⁵ ) as an active ingredient. for animals, except for humans.

Description of the Figures

FIG. 1 is a photograph, taken from an electron microscope, of the new bacteriophage ’> C2sL> <ΜΠ '<ΜΡ óe> ο Ά'ocromip ιΛ> 4K'

FIG. 2 shows the result of a pulsed field gel electrophoresis (PFGE) of the new bacteriophage <& CJ22.

FIG. 3 illustrates the result of a polyacrylamide-dodecyl sulfate gel electrophoresis

6/28 sodium (SDS-PAGE) of bacteriophage <3> CJ22.

FIG, 4 is a graph showing the result of an acid resistance test of the new bacteriophage Φ (1Ι22.

FIG. 5 is a graph showing the result of a heat resistance test for the new bacteriophage ΦΟ22.

FIG. 6 is a graph showing the result of a test. drought resistance of the new bacteriophage ΦΟ22.

IdeaI co »fig» ration

In the following, the present invention will be described in detail. Obvious and easily interpretable considerations by those skilled in the art will be omitted in this specification.

In a general aspect, the present invention provides a new bacteriophage of> CJ22 (KCCM11364P), with a specific bactericidal activity against small intestine (CP).

Is it known that Ctortrit / iM ?? perfiifígemr which is a large gram-positive mandatory artaerobic bacillus, has no spines and forms a spore. CZmwíZiíw peryHngens, which is a bacterium that causes diarrhea or similar problems in animals, particularly in domestic animals such as poultry, pigs and the like, has been recognized as one of the dangerous and fetal pathogenic bacteria in the animal industry such as / i / fig-igeha, causing avian typhoid fever.

A bacteriophage is a bacterium-specific virus that infects specific bacteria to suppress and inhibit the growth of the bacterium, and constitutes a virus containing dgsoxyribonudeic acid (DNA) in single or double strand, or ribonucleic acid (SNA) as a genetic material.

() bacteriophage <1> CJ22 according to the present invention, which is a specific bacteriophage to selectively infect C Vwridíw? per / íwgem, has an isometric capsid

7/28 and a tail that does not contract and, morphologically, belongs to Sójómvmáíe (FIG. I). Data for nucleic acid sequence homology analysis between bacteriophage ΦΟ22 and other bacteriophages are shown in Table 1. The bacteriophage '$ CJ22 activity remained stable for 2 hours in the pH 4 to pH 9J range (acid resistance, see FIG. 4).

Ο Φ € .Ι22 retained its activity for 2 hours when it was exposed to 60 * C (heat resistance, see FIG. 5), and its titre was reduced by 2 log after drying (see FIG. 6). The sequence of nueleic acid of bacteriophage <t> CJ22 is the same as SEQ ID NO: 1.

The bacteriophage <t * CJ22 isolated for the first time by the inventors of the present invention was deposited at the Korean Center for Culture of Microorganisms (361-221, 10 Hongjedong, Seodaemun-gu, Seoul, Cot eia) under deposit number KCCM11364? on January 30 - 2013,

In another general aspect, the present invention provides a composition for the prevention or treatment of infectious diseases caused by OartrÁâw? per / róTgeos containing the bacieriotugo $ CJ22 as an active ingredient. As a preferred example of the composition, the present invention provides an antibiotic.

Since the bacteriophage ΦΟ.Ι22 has an antibacterial activity capable of specifically killing Qo.ww </ õ », the bacteriophage ΦΟ322 can be used to prevent or treat diseases caused by the infection of perines. A typical example of an infectious disease caused by (J / osvrW? Wu per / rmgímv that can be treated with the use of bacteriophage Φ022, is the neurotic entity, but the present invention is not limited to its treatment.

Entente necrotica, which is one of the main infectious diseases caused by GasírAm per / rmgcm, is characterized by being a bacterial disease whose most frequent occurrence occurs in domestic animals, particularly domestic birds, causing 25 significant damages. The disease can attack poultry, I broke chickens in

8/28 all ages, but it manifests mainly in chickens (2 to 5 weeks of age). Raised free and also frequently in chickens (.1 to 16 weeks of age) reared in cages, 'As -Cl & stridíum pefpingens è excess! proliferating in the small intestine, symptoms of necrotic enteritis occur, causing necrosis of the gastrointestinal mucosa, sudden diarrhea, and similar effects. For example, pigs with very acute necrotic enteritis die after 1 to 2 days of occurrence, and in the case of acute necrotic enteritis followed by bloody diarrhea, the animals die after 2 to 3, .In addition, in the case of Sub-necrotic enteritis acute, diarrhea (without blood in the stool) continues for 5 to 7 days and then weakness and dehydration occur, and in the case of necrotic enteritis. chronic, intermittent diarrhea that can cause growth disorder.

The term '' prevention, as used in this descriptive report, refers to all actions related to the application of bacteriophage ΦΟ22 and / or the composition containing bacteriophage ΦΟ22 as an active ingredient for animals, except for humans, in order to to suppress the corresponding disease or delay the occurrence of the disease.

() term '' treatment, as used in this specification, refers to all actions related to the application of bacteriophage OCJ22 and / or the composition containing bacteriophage ΦΌ22 as an active ingredient for animals, except for humans, for allow improvement or relief of the symptoms of the disease caused by the infection,

An example of an infectious disease caused by which bacteriophage Φ € '122 and / or the composition containing bacteriophage <1> CJ22 .as an active ingredient can be applied, is necrotic enteritis, but -the present invention is not limited to it .

The composition for the. prevention or treatment of infectious diseases caused by CWvÁÚw according to the present invention, may contain the bacteriophage

9/28

ΦΟ22 with a preferable content of 5xUF to 5x10 ' ³ pfu / ®fo, more preferably, 1 x 1.0 ”a. 1x10 '·' · 'ptufofo

The composition for the prevention of the treatment of infectious diseases caused by CAxs / nJhuu piírpfngtífj * in accordance with the present invention can also contain an acceptable pharmaceutical excipient and be formulated together with the excipient to thereby be supplied as a food, a drug , a food additive, an additive for drinking water and the like, The term acceptable pharmaceutical excipient ”, as used in this specification, means an excipient or a diuent that does not stimulate the living organism and does not inhibit the biological activity and properties of a administered composition.

.10 The type of excipient that can be used in the present invention is not limited to a specific type, and any substance, commonly used in the art as an acceptable pharmaceutical excipient, can be used. As non-restrictive examples of the excipient, we can mention common saline solution, sterile water, buffered saline, Ringer's solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. One or a mixture of at least two of these ingredients can be used.

In addition, if necessary, other additives in general, such as an antioxidant, a buffer solution, a bacteriostatic agent, and / or the like, can also be added or used, and the composition can be prepared with. an injection formulation, such as aqueous solution, suspension, emulsion, or the like, pills. capsules, granules. tablets, or the like, by adding a diluent, a dispersant, a surfactant, a binder, a lubricant, and the like, to then be used.

A method of administering the composition for the prevention or treatment of infectious diseases by Cfosíráfíum per / ria has no limitations, so that any method commonly used in arté can be used. As an example,

10/28 limiting the method of administration, the composition can be administered orally or parenterally.

As non-restrictive examples- of the formulation for oral administration, we can cite troches, lozenges, tablets, suspensions.-aquosss, oily suspensions, prepared powder, prepared granules, emulsions, hard capsules, soft capsules, syrups, elixirs or similar.

In order to formulate the composition according to the present invention in a formulation such as a tablet, a capsule, or the like, the formulation may also contain a binder such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, 10 cellulose, gelatin; an excipient such as dicalcium phosphate, or the like: a disintegrant such as corn starch, sweet potato starch, or the like; a lubricant such as magnesium stearate, calcium stearate, stear! I sodium graph, polyethylene glycol wax, or the like. In the case of capsule formulation, the formulation may also contain a liquid excipient, in addition to the materials mentioned above.

As a method of parenteral administration, it is possible to use an intravenous method of administration, an intraperitoneal method of administration, an intramuscular method of administration, a method of subcutaneous administration, a method of local administration, etc. In addition, it is also It is possible to use a method of applying or spraying the composition on the disease site, although the present invention is not limited to such a method.

Examples of formulations for parenteral administration may include injectable formulations to be applied by injection, subcutaneous, intravenous injection, intramuscular injection, or the like; suppository formulations; spray formulations, such as aerosol formulations, which can be inhaled through the respiratory system, 25 or the like, although the present invention is not limited to such formulations. In order to

11/28 preparing the composition for the injection formulation, the composition according to the present invention can be mixed with a stabilizer or a buffer solution in water to prepare a solution or suspension and then a. prepared solution or suspension can be formulated in a single dose into an ampoule or fiasco. In the case of the formulated spray composition, such as the aerosol formulation or the like, a propellant or the like can be mixed together with an additive so that the composition condensed in water or powder is dispersed.

An appropriate application, spray, or dose of administration of the composition for the prevention or treatment of infectious diseases by Cfatâríítíufn per / Hngíms can be differently determined depending on factors such as age, weight, sex, degree of symptoms of the disease, type of disease. feed and excretion rate of animals subject to administration, or similar conditions, as well as the method of formulating the composition, the method of administration, the time of administration and / or route of administration. In general, a veterinarian with knowledge in the art can easily determine and prescribe an effective dose for the intended treatment.

In another general aspect, the present invention can provide an antibiotic containing the bacteriophage ΦΟ22 as an active ingredient,

The term antibiotic, as used in this specification, means an agent capable of being administered to animals, including humans, in the form of a drug 20 to kill bacteria, and corresponds to a concept collectively representing a preservative, a disinfectant, and an antibacterial agent.

The antibiotic containing the bacteriophage ΦΟ22 as an active ingredient, according to the present invention, may have a high specificity for pc.fortegcnx compared to other antibiotics available in the prior art, with the ability to not kill beneficial bacteria, but only specific pathogenic bacteria.

12/28 being still characterized by not inducing resistance to the drug, so that the antibiotic according to the present invention can be supplied as a new antibiotic with a longer life span when compared to the antibiotics available in the technical state.

In another general aspect, the present invention can provide a food additive and an additive for drinking water containing the "bacteriophage"> CJ22 as an active ingredient.

The food additive and the drinking water additive according to the present invention can be used in such a way that the bacteriophage ΦΟ22 or the composition containing it is individually prepared as a food additive or for drinking water, and then mixed for food or drinking water, or in such a way that the bacteriophage 10 ΦΟ22 or the composition containing the bacteriophage <IX, J22 - is added daily when preparing the food or drinking water.

The bacteriophage foCJ22 or the composition containing the bacteriophage Φ ('122 used as a food additive or for drinking water according to the present invention can be formulated in liquid or solid state, preferably in powder form.

Drying method for the preparation of the food additive and the drinking water additive under. The dry powder form according to the present invention has no limitations and a method commonly used by those skilled in the art can be used. As non-limiting examples of the drying method, we can use a natural air drying method, a natural drying method, a spray drying method, a freeze drying method, or similar methods, one of these methods can be ui.it used alone or at least two of these methods can be used together.

Another non-pathogenic microbe can be added to the food additive or drinking water additive »As a non-restrictive example of the microbe to be added, it is possible to choose a microbe selected from a group comprising Boei / fos sp ... capable of producing, protease, lipase and / or sugar converting enzyme, such as Brrefows fofoo / ís or

Similar 13/28; a vp. with physiological activity and degradation activity for an organic material under anaerobic conditions, such as the cow's stomach; mold fungus capable of increasing the weight of a domestic animal, the production of milk, and the digestion of foods such as Aspergí / iw e ^ w, or similar; and yeasts such as 5 & ce /? í? romj ^f Ci <v tvreràw, or the like. One or a combination of at least two of these microbes can be used,

The food additive or additive for drinking water containing the bacteriophage ΦΟΙ22 as an active ingredient, according to the present invention, may also contain other additives, as necessary. As a. non-restrictive example of additive, it is possible to use a binder, an emalsificant, a preservative, and the like, which are added to prevent deterioration of the food or water; amino acids, vitamins, enzymes, probiotics, flavoring agents, nitrogen-free compositions, silicates, buffer solutions, coloring agents, © xtracting agents, oligosaccharides. and the like, which are added in order to increase the usefulness of food or drinking water. The additive may further include a food mixing agent, or the like. One or a combination of at least two of these additives can be used.

The food additive can be added in a content of 0.05 to 10, preferably 0.1 to 2 parles by weight. based on 100 parts by weight of the food. The potable water additive can be added in a content between 0.0001 to 0.01, preferably from 0.001 to 0.005 2 (1 parts by weight based on 100 parts by weight of drinking water. The activity of the bacteriophage d> CJ22 against CfoxfràZwn per / r / ngens can be sufficiently demonstrated in the above mentioned ranges.

In another general aspect, the present invention provides a food or drinking water prepared by the addition of a food additive or drinking water containing the bacteriophage ®CJ22 as an active ingredient, or by the direct addition of the bacteriophage ¢ (5,122 ..

14/28

The food used in the present invention has no limitations, so that any other food commonly used by those skilled in the art can be used. A non-restrictive example of food may include plant foods such as grains, roots and fruits, - food processing by-products, algae, fiber, pharmaceutical by-products, fats, starches, eubitocytes or cereal by-products; and animal feed, such as protein. inorganic materials, fats, minerals, proteins from individual cells, animal plankton or food. One or a combination of at least two of these -foods can be used,

The drinking water used in the present invention has no limitations, so that any drinking water can be used in the present invention.

In another general aspect, the present invention can provide a disinfectant or cleaning product containing bacteriophage ΦΟ22 as an active ingredient. A disinfectant or cleaning product formulation has no limitations, regardless of whether the disinfectant or the product cleaning agents can be prepared with any formulation known in the art.

Can the disinfectant be sprayed to eliminate Oosrr / dhi? » / jer / ringmy in a region where animals live, in a slaughterhouse, in a mortality area, in an o.u kitchen in one. kitchen equipment, or the like, the present invention is not limited to these examples. () cleaning product can be used to wash the skin surfaces or all the locations of the bodies of animals exposed or to be exposed to CZostrW / u »? / «/ Glargtouv particularly poultry or swine, the present invention being not limited to these examples.

In another general aspect, the present invention provides a method of preventing or binding infectious diseases caused by Cfaimfam pez / rrgem, using it

15/28 bacleriophage 0CJ22 or the composition containing the bacteriophage Φ € 522 as an active ingredient. The infectious disease may preferably be necrotic enteritis, the present invention being not limited to the treatment and prevention of this disease. The objective of preventing or treating infectious diseases caused by Cfr.s / rtóZZum perfrfogem 'can be applied to poultry or pigs, the present invention is not limited to the treatment of these animals.

Specifically, the method of preventing or treating infectious diseases in accordance with the present invention may include administration of the bacteriophage ΦΟ.Ι22 -or the composition containing the bacteriophage ΦΟ22 as an active ingredient for infected animals or those in. risk of infection by tfessíriífem® in an effective dosage f • macéuuca, except for humans. It will be apparent to those skilled in the art that when the pharmaceutical composition is administered to the patient, the appropriate total daily dose can be determined by an attending physician or veterinarian in accordance with his medical judgment.

A specific pharmaceutically effective dose of the bacteriophage ΦΟ22 or the composition containing the bacteriophage ΦΟ22 as an active ingredient for a given animal, can be determined by considering the time of administration and the route of administration of the bacteriophage $ CJ22 or the composition containing the bacteriophage Φ <022, the rate of secretion of the composition, the duration of treatment, or similar factors, added to the type and degree of response desired ^ age, weight ^ general health status, sex, or 20 diets of a certain animal. In addition, the pharmaceutically effective dose can be changed according to several factors, such as the ingredients of the drugs or. other compositions. used simultaneously or separately, and other similar factors well known in the medical field.

The bacteriophage Φ022 according to the present invention or the composition containing bacteriophage ΦΟ22 as an active ingredient can be administered as a .16 / 28 pharmaceutical form (nasal spray) for animals or administered as a method of direct addition to a food or in the drinking water of the animals, which then eat the food or drink the drinking water. In addition, the l22 bacterlophagus or the composition containing the same can be mixed in a food or drinking water in the form of a food additive or additive for drinking water, to then be administered.

The route of administration and method of administration of the bacteriophage <ÍCJ22 according to the present invention or of the composition containing the bacteriophage <PCJ22 as an active ingredient are not limited, so that any route of administration and method of administration can be used, provided that the bacteriophage ΦΟ22 or the composition eating the same can reach the desired tissue, that is, the bacteriophage ΦΟ22 or the composition containing the bacteriophage ΦΟ22 as the active ingredient - can be administered via various oral or parenteral routes. Non-restrictive examples of the route of administration may be oral, rectal, local, intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous and nasal or inhalation administration, or the like.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are illustrative only and are not intended to limit the scope of the present invention.

[Example 1] Isolation of bacteriophage infecting C / wrâZfom <Example l-I>

Sçlç £ ãndobgçt £ | i ^

A sample of 5 (W of feces collected from the Samwhaw Gps company was isolated. Breeding Agri. Inc., A chicken and pig farm in the southern Chtmgchong province. Republic of Korea, Inserted in a bottle. Centrifugal and centrifuged at 4,000 rpm for 10 minutes, the supernatant was filtered with a 0.45pm filter to prepare a sample solution, and then a soft water overlay method was applied

I 7 3 using the prepared sample solution. The Soft Agar Overlay method is a method of observing the action of bacterial phage lyses using host cells growing on surface agar (on a solid medium with 0.7% agar).

Specifically, ISwú of filtered sample was mixed with 150fo? of a stirred solution of the culture (OD ^ ^:::: 2) of CZosrrà / fton pe / Yrfogens, (CP. BCCP 17-1) isolated at the local Animal and Plant Quarantine Agency, and 2W ^? infusion of IOXBrain-heart (hereinafter referred to as' ΒΗΓ medium) (composed to generate a final volume of 11.) and grown at 37X for 18 hours. Then, the culture solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 «» filter. Then, a mixture of .5 '.0 agar at 0.7% (w / v) and 15ÓM of the stirred culture solution (ODe.' ~ 2) of Cfosfoáfow / ether / rrâgmr (B-CCP 17-1 ) was poured and hardened on a BHI plate (BHI-t 0.2% sheep s-anguish), i (W of the filtered culture solution of the sample was poured over it, followed by a culture of 3 ( for 18 hours, then a plaque was formed.

Once filtered, a. culture solution of the sample, in which the lysis was generated, was adequately diluted and mixed with 150fo ^; of agitated culture solution (OI) ^ »~ - 2) of êfoztírfoíwj Pérfofogws (BCCP 17-1), the overlay method on soft agar was performed, thus obtaining a single plate. Considering that a single plate was formed to panic is a single bacteriophage. a single plate was selected to purify and isolate the single bacteriophage, placed in 400 /./ 0 of an SM solution (NaCI 5.8 g / 1;

MgSO47H2O 2g / I; 1 M Tris-CI (pH 7.5), 5 µm H2O, composed in order to generate a final volume of IL), and left at room temperature for 4 hours, thus purifying and isolating a single bacteriophage.

In order to ensure a large quantity of the isolated bacteriophage, 100 µl of a supernatant from a single bacteriophage solution was selected and mixed with

18/28

12 month of 0.7% agar and 500x2 of the stirred solution, from the Clostridium per / rfogem culture (BCCP 17-1), followed by the method of overlaying soft agar in a LB medium with a diameter of 150 mm. After pouring the ISw solution? of the SM solution on a plate in which lysis was completely generated, the plate was gently stirred at room temperature for 4 hours, thus discharging the bacteriophage on the surface agar. The SM solution. in which the bacteriophage was discharged was recovered, with chloroform being added in an amount of 1% of the final volume and adequately mixed for 10 minutes, followed by centrifugation at 4,000 rpm for 10 minutes. The supernatant obtained as described above was filtered with a 0.45um filter and stored at .10 cold temperature.

<Examples í-2>

Large-scale culture and purification of bacteriophage

The selected baeteriotago was grown on a large scale using CAuvòVfomz? ner / mígem (BCCP 17-1) and then the bacteriophage was purified.

Specifically, I% of the stirred solution of the per / Hpgen culture,? (BCCP

17-1) was inoculated in a liquid culture medium for mass production and, at the same time, the baeteriototh was placed inside it in a multiplicity of infection (MOI) of 0.1, simultaneously with the inoculation of Ç / osírÀ / Orm pcq / wtgms (BCCP Π-l), thus performing co-infection. Then, the static culture was performed at 3 ° C under 20 anaerobic conditions.

Then, the centrifugation was performed at 4 ·% and .12,000 rpm for 20 minutes, and then the supernatant was filtered with a 0.45 / zm filter. Then, NaCl and polyethylene glycol (PEG) were added to the supernatant filtered so as to obtain a final concentration of IM and 10% (w / v) respectively, and the mixture was left at a temperature of 4 ^and C for 8 hours or more. Then, a centrifugation was performed.

19/28

4 ^to C and 12.00 () rprn for 20 minutes, the supernatant lbs removed and precipitates were obtained.

The obtained precipitate was resuspended in the SM solution and left at room temperature for 20 minutes. Then. the supernatant was filtered with a 0.45 µ filter. and an ultracentrifagation (35,000 rpm, 1 hour, 4 ^and C) was performed using a glycerol density gradient method (density: 40%, 5% .glycerol), thus purifying the bacteriophage Φ € Ι22, after 4> Purified CJ22 is resuspended in 500fo ^; of the SM solution, a titer was measured.

The inventors of the present invention named the bacteriophage obtained by extracting the stool sample with specific bactericidal activity against the '' Bacteriophage 4> CJ22 ”, and deposited the bacteriophage in the Korean Culture Center for Microorganisms (361-221, Hongjedong, Seodaemun-gu. Seoul, Korea) on 30. January 2013 under deposit number KCCMI1364P.

<Example 2>

Morphological observation of gl22

The purified ΦΟ22 bacteriophage was diluted in 0.01% gelatin- solution, and then fixed in 2.5% glutaraldehyde solution. The fixed bacteriophage was poured onto a carbon-coated mica plate (about 2.5 mm x 2.5 mm) for 10 minutes and washed with sterile distilled water. A carbon film was placed on a copper grid, tinted with 4% uranyl acetate for 30 to 60 seconds, dried, and observed through a microscope. electron transmission (JEM-I0II, 80kV. magnification: XI 20,000 to X200.00Ü) (FlG.l).

FIG. 1 shows a photograph of the bacteriophage foCJ22 made by the electron microscope where it is possible to observe that, as the bacteriophage does not have an isometric capsid and an eontractable tail, the bacteriophage belongs morphologically to <Example 3>

Anahge do tgnranhq dq genomic DNA from ΦΟ.Ι22

Genomic DNA was extracted from the bacteriophage Φ € .Ι22 purified by uhracentrifugation. Specifically, ethylenediaminetetra.cetic acid (EDTA, pH 8.0}, proteinase K and sodium dodecyl sulfate (SDS) were added to a solution- of the purified bacteriophage culture ΦΟ22 in order to have a final concentration of 20 mM , 50μ /%, and 0.5% (w / v) respectfully and then were left at 50 ° C for 1 hour, after which an equal volume of phenol (pFI 8.0) was added, stirred if, and centrifuged at room temperature and 12,000 rpm for 10 minutes, thus obtaining a supernatant.

The supernatant was mixed with an equal volume of PC (phenol: chloroform ^::: 1: 1) and centrifuged at room temperature- and 12,000 'rpm for 10 minutes, thus obtaining a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at room temperature and 12,000 rpm for 10 minutes, thus obtaining a supernatant. The supernatant obtained was mixed sequentially with 10% (v / v) of 3.M sodium acetate and a double volume of cold 95% ethanol based on the total volume, and left at -20 ^to C for 1 hour. Then centrifugation was performed at 0 ° C and 12,000 rpm for 10 minutes, and the precipitate was obtained by removing the supernatant. Then 50:% Tris-EDT / A (TE) buffer solution (pH 8.0) was added to it, to thereby dissolve the precipitate obtained. The extracted DNA was diluted 10 times and the concentration was measured by measuring the absorption in ODs ^.

Then, DNA was loaded in 1% electrophoresis in pulsed field gel (F.PFGE), in agarose gel, and electrophoresis was performed at room temperature for 20 hours using a program from the BIORAD PFGE system, program 7 (size: 25-100 kh; switching time: 0.4-2.0 seconds, linear; direct voltage: 180V; reverse voltage 2-5: 120V) (FIG. 2).

21/28

FIG. 2 is a photograph of the pulsed field gel electrofbresis (PFGE) of the genomic DN-A of the bacteriophage foCJ22, where it can be confirmed that the genomic DNA of the '4> CJ22 baeteriophage has a size of about 56kb.

<Example 4>

Anáhe du paprlo d

15/2 of the purified eter22 baeteriole solution (titer 10 ^ib pfu / ml) was mixed with 3 x 2 of a 5.X SDS sample solution and heated for 5 minutes. Then, the bacteriophage total protein Φ (?. Ι22 was expanded in 15% SDS-PAG.E gel, and then the gel was stained at room temperature for 1 hour using a coomassie blue dye solution ( FIG 3).

FIG. 3 is a photograph of the electrofbresis showing the result of the SDS-PAGE performed on bacteriophage ΦΟ22, with main proteins and sizes of about 40kJ) at, 5IkDa, 53kDa and 70kDa being observed. In FIG. 3. It's a protein that becomes, a standard for measuring molecular weight, <Example 5>

Aná [se € 322 gene sequencing is € 322

In order to confirm the genetic characteristics of the purified bacteriophage Φ <322, the DMA of bacteriophage ΦΟ22 was analyzed using a titanium FLX sequencer (Roche), which is a gene analysis device. Genes were assembled at Maerogen INC. using GS and the new software (Roche ·.), A. analysis of the sequence of an open reading frame was performed using Gene.MArk.hmm, Glimmer v3.02, and FGENESB software. The identification of the open reading frame was carried out using BLAS1P and they pro- moted the Pro Scan interface.

The sequence of the gnome of the new bacteriophage obtained showed several similarities with those of the existing baeterioíagos, but it was confirmed that a bacteriophage with all fractions (100%) equal to the bacteriophage of the present invention - did not exist. In this way, it was confirmed that a new bacteriophage-according to the present invention was isolated.

Data from the homologization analysis of the nucleic acid sequence among the bacteriophage

ΦΟ22 and other bacteriophages are shown in table 1, (Table 1]

Fergwsia ObíW> tosusçsís È-Vaiue Etefiíitoks <? o «spri mcnía End .to!. :: · .t .-> í: i · X-i .. 1%) cosíigftWfíf, p? 730íí? 3 44! 31 344 Puuib protein »: Aqusfes VFSj 317-: 4 53; '!? 2 34 <00 1 715 Ρϊί> ί't «ít for <forft!:> C. female 55®; 1 Áryíwid:; S-xniiitexig / eii: 1 44441 1 & 41 141/3 i 5 32 .................. 44: sis30f81í, jpC> 33f 1 374 <7 345 Hyposienic proteins A3ÍA4XS. 43344 1.3 ::.: .-: AftslxsrííiR: ·! (i.) i> SY? 3Xq 7E-47 41 _ there already>. 2H68 24 i íl 2X57 Pwjtffe / í; <· ;? AC 3 2545 IftStri jAílil píí: · ίήν: ^ ΐ) ίΐ $: D.. JGWÍS7 7E-55 / 43/154 AT 2" 1 2nd? S.:.:.·:...·::1 FARB llArísM <: g * (CtoüiáiíiS! P%! BSÍAS C At ' _: .4': 1433: 54'22 4?; »9 t-X e: iHSg34441 .ísj-OTÍÇ 533? 28 ! 254 PíiXíS: <S de (fefíS:! - fe there: i:; -> lf 1 «'1: 1 ::::: í :: T: ^; > · (. <: Fs' 0. 1 SÍ? 3 : j IS-IS? 2443421 47 s: S4t: g44441>; rs? í> í7 ! W 28 5? 5 JWteiaa Sipí / iiLea -WRaI - 7434 ALEX [SuMetenacesí! fsicfeiMW'i C4431 43-14 Wis-S A:> <- «Hi: g4444i wffiÓOs ·) 134? 583 753 pAlií: iü <: hípt.íêí: A:! ^! CC7434,55i4 K yaüi-tiKss sp>;<. 7424J 13-45 3175? 54 iftVígíOuOi ísrO Si 14 50 ’ • 1'4 / iWwíiffis iiijWli & i C4.J: '·; - 14 í: Ji;.:' :: 4νί ;; 1ι «·,: Ρ · Bái sy. 657: ‘5 & Í2 in 73 «I: iisg4724i, & - ®03! W7 Wó Ajuieins mpeteiiea 24: 4 ::: - 3: 1 ::. 1: 447 444: 3 s ixwie-rwfàg: · nôí> tóentifwadot 53-41 :: 44/374 33 Si: K-> g44áll JXÍ44422 742 7 4: 7 ZkdP ijAwiiX ;: sp. IS1 41514 53 /:? - ' 5x eçntigiiO® i j wiíX® i 5 5S4 Ϊ sss : 7? :: l · ·: i!: · ..: llillílWjVSíia ICiiAidtois ptohiissws4- sV. XllSiMíi 43-75 77 /! 48 5A

eWSSÜW .: i jerUM 8 Í059 28 5047 p :: L :: f Víj FB8. \ P: ’<XC8'.3 pA ': ph $ ig.e ί &: i- lu.s sw6 $ ^. GUb · i! 58 str. 868 .: 43-52 5: 5334 5 45) 8 3M I'BsX Proteins M45 · • CiosSrioium «berouxJtem A'UX 2 73:« · ÍE-Í4 48.:422 9 ·? x -. i l \>> 403 • δ 403 Pretaíná AtpótéEaá r «: MOBEJ: Aí5õ I'Runawcaceus oSwm ATCC 29:74] 5: .- (O 442 39 33 2502 25 1239 .Proteins taii tape. iàmíJia TPW ': iWerpetosipium í: òr-sf4 :: j> -sK USM? S3’J 2E-S2 5S33359 47 í: i>ssixixXKK; i! <Μí _: $! 5> -> 43 323 Prj ^ sps hspotó-sca U \ i V>) UR '.: I 33-½ {Bacstiiks sp í NLA3E] i:: -45 87/7 52 eootígOòOÒi í: t®037 ............ 32: 5 : 9 .875 # teKftófego Mb .proteins F (Geobaeilsus rherirppAUUioSrfe TM) ~ 33,320: 26-SY: 5] 47334 ctmt: $ 000 i ..p: 790928 412 U! 42 ·: hoteirta hippo-Aisa RÍJVO5E. 3: 333: E :::: ·.?: · .A7ÇC 29:74] 8 U- í <6 473: .30 i: 00Kgõ * Hi • us .:. 333 P: :::; hipopXeX y:;:. a: .ivs PBSX: Hr :: :::: · .; : .4: prWiSOÍSriS Xri7} í P-is? 3775 53 3S CAF: £ 6600 í _õ'1 '; <8U6a5 Ϋ>: ϊ : t> 882 P; vtfeiDa h :: *> i. <frog: í <.- st <\> K> í? rvyíJ; i Í'G-í <uív '> f.rV ’. ::: S: .íí? · ^: -38 ^ 4.5: -24 ! 6: r «9í 55 .W’M ^ XWi-jSrRWW 2SÍ .: : Orofeiiis òipowiíçs RUMOBA.ORSJ i riunnntieoseas ofesirm Λ i:.:. 29574; ': ÀÍ'6.> 554/395 32 νοηίΐΰ, ΟΰΓίΐνί _5 <g <X) ü25 ^: 426 Ssi 4: 7 Htpotèt protein: ca3SSCS .. 30353 qUsSuSíUiiiis sttbsp. :>: 3b: j: ss Sir. S33-3] 35: -: 3 463 S i 3 · -: OOfijigCS-sX? i orSVsOo 363 3 . >> < Usfetes hipíUÜÍi mXX.MtmRAÍT .3747 [.asíeilriá Sp. Ί XÍ.ArÇ: 6-E-13 45757: 2 44 <>: ϊϊ <ύ <>, ΐ · ΐ) 0ν1 o: '40i; 02? : 332 : : 32 « PreKirsa .taiis <hesth; i> rs. <:: r:> sporí> s :: -:;: yatmgkie Ο8Μ! 7734] : 5: <' 10-2449 37 irxxsmrwl «rOAO? : -03- 3432 jéMeíriír.sÕBííéíAa üiAotòOíM .54926 jBaciiias í :::. I: I3SÍí ::> $ ÍS:.: .- 0:. :: · stf. 304: 23] "-/-Hi: 4: 'e <> n6g ^{ X8XP, ^ er ^ M-í <78 í 266 dC.M-P ii «srr: iMs <r, putama íÂrcirseogiosBS fuípsíos: ’93 4334] 5E-23 6) 3:34 4: 5

24/28

25/28

The partial sequence of the genome of bacteriophage oma22 was found to be the same for SEQ ID'Notl. The genome sequence was determined using a genetic analyzer.

«Example 6>

De22 stability test-depending on pH

In order to confirm the stability of bacteriophage ΦΟ23 in a low pH environment. the stability test was carried out over a wide pH range. (pH 4.0 / .5.5 / 6.4, 6.9 / 7.4 / 8.2 / 9.0 and 9.8).

For the test, several pH solutions [sodium acetate buffer solution (pH 4.0, pH 10 5.5 and pH 6.4), sodium phosphate buffer solution (pH 6.9 and pH 7.4), and a Tris1-ICI solution (pH 8.2 / pH 9.0 and pH 9.8)] were prepared at a concentration of 0.2 M, respectively.

After 90 / ίέ of each of the pH solutions was mixed with a bacteriophage solution with a title I .OXHfpfo / W, such that a concentration of each 15 pH solution became 1 M, each of the solutions pH was left at room temperature for 30 minutes, 1 hour, and 2 hours. Then, the reaction solution was diluted step by step. the diluted solution in each step was poured ^u and cultured at 30 C for 18 hours using an overlapping soft agar method, and the title TBI measured by da.presença or absence of lysis (FIG. 4).

A. FIG. 4 shows a result of the $ € J22 bacteriophage acid resistance test.

As illustrated in FIG. 4, it was confirmed that the bacteriophage ΦΟ22 does not lose its activity and remained significantly stable, in a pH range of 4.0 to 9.8 for up to 2 h-sma.

«Example 7>

Tesredemabilidadedu.M ^ Áldepender ^ J ^

26/28

A test was carried out to confirm the stability against the heat generated during the bacteriophage formulation process, in the case of using the bacteriophage as a food additive formulation between the bacteriophage formulations,

Specifically, 20µW of the bacteriophage solution ΦΟ22 with a title 5 I.OXHfipfoW was left at 60 ^to C for 0, 10, 30, 60, and 120 minutes. Then, the solutions above were diluted step by step, KW of each of the diluted solutions- were poured and grown at 30 ° € for 18 hours using a soft agar overlay method, and the titer was measured by the presence or absence of Use (IIG, 5).

FIG. 5- shows the result obtained through- one. heat resistance test of bacteriogenic à22 .. As shown in FIG. 5, it is possible to verify that the activity was not significantly decreased until the bacteriophage ΦΟ22 was exposed to 60 '% for 3 hours.

<Example 8>

Stability test of the 44322 against the seqegm A test was carried out to confirm the stability against drying conditions 1.5 generated during a bacteriophage formulation process, in the case of using the bacteriophage as a food additive formulation among the other formulations of the bacteriophage.

Specifically, X22 bacteriophage solution KXW. titmo with a 1.0X II) ^are pfoW -secos were 60% for 120 minutes, using a vacuum centrifuge concentrator (Speed * V.accum concentrator 5301, fcppendorf). The approval obtained after 20 a. drying was placed and resuspended - in an SM solution at a value equivalent to that of an initial solution at 4 ° C for one day.

Then, the above solutions were diluted step by step, the solution of the solution diluted in each step was poured and cultivated at 30 '% for 18 hours by the soft-water overlap method, and the titer was measured by the presence or absence of lysis (FIG. b).

FIG. 6 shows the result of a drying resistance test for bacteriophage ΦΟ22. As illustrated in FIG. 6, it is possible to notice that the bacteriophage OCJ22 was reduced by about 2 log after drying.

«Example 9>

Spectrum test of dofoCJ22 infection with respect to these JalfooádyaggmJg 'Whether or not it has lytic activity. bacteriophage ΦΟ22 was tested on 45 wild-type strains of C & m7fú: / íwj isolated by the Animal and Animal Quarantine Agency and Kunkuk University other than C / tó'tr «/? mn / wforçgmv (BCCP 17-1) used 10 no ex perirn en to.

Especificaeftte, UW of bacteriophage solution ΦΟ2.2 with a title L0X10 ^K 'pfo / 8 $ were mixed with a 150 foot solution ^; of agitated culture (OIXw, · ^::: 2) decade one of the strains, being poured and cultivated for lb hours by the overlay methane in soft agar. Then, it was observed whether a plaque was formed or not.

As a result of the experiment, among 45 strains of wild type, oe. Cfetrafoen per / rfogemç 4-2 strains were infected, in such a way that the proportion of infection was about 93.3% and the lysis ratio was about 75.6%. The results are shown in Tables 2 and 3.

[Table 2]

Origin of CP

K unkuk Universitv

University

bforpe d · »ϊ T Infectivity HI 3 b ·; .................................................. . TlLYS-3 JSH-i ·· KCCM 40947 ^! -% ‘r-f · K.íW-2 · Ϊ · - ··> ·

kt i M12098

28/28 [Table 3]

Origin of ÇP .surge from t, P In feet A jj.uk- Okay, okay? CP Infecdv age Agi'Bciu de Onarentena Vegetable and Animal CP-KJW-I B ((P43-I 4 / - i Animal and Plant P-1 \? U .5.4.4, B (’CP4-M +: Quaramme Agency 2P 03 x 2 H-r BCÍ P P-2 r ++ CP-BC-1 k-H · B (LP4S-3 .44., 4 CP-BSW-4 H->! BCCP50-1-3 + 7- CPdiBM-2 - ++ BCCP50-1-8 UP Hl. 4, .4.4, B <(PM-J-l UP-KW-I 4..4 .., BCCP5I-O5 CP-BS-1 B (<P52-2-8 CP-HL-I 4..44. BCCP53-2-3 CP-UN-1 BCCP54-3-8 .............................................. BCCP17-I 4.4.4 BCCP55-34 -r-t-À- Hi i p? Xt 4..4.:. SIH t P42.O-2 44., 4. B (CPU-2 SBICPOJ 4.4.4, JU {p ^ -i -y-k -x · SIR (Pte ' BCCP39-I SBCCP361 Λ., Λ, .ζ. \ 1> BCCP40-1 ÍÇ- ELCCP Suksan Kim χ, -4 ·· BCCP4I - 3 4.4.4. EI.CCP6-I Í niesii rms Η * BCUP12-2 .44.4 .. Hit P6! appendage

κι ί \ · ιχηκ \ çòi s

Claims (8)

1. A new bacteriophage 4> CJ22 (KCCM11364P). characterized by having a specific bactericidal activity against Cfottrídfam pez / r / ngenr. 2. A composition for the prevention or treatment of infectious diseases caused by Cbstridium characterized by comprising the bacteriophage ΦΟ.Ι22 (KCCM11364P) of claim I as an active ingredient. 3. The composition according to claim 2, and supported by the fact that the infectious disease is necrotic enteritis. An antibiotic, characterized in that it comprises the bacteriophage · <1> CJ22 (KCCMl 1364P) of claim 1 as an active ingredient. 5. A food additive or additive for drinking water, characterized in that it comprises the bacteriophage Φ € 222 (KCCMl 1364P) of claim 1 as an active ingredient. 6. A disinfectant or cleaning product, characterized in that it comprises the bacteriophage Φ (? 322 (KCCMl 1364P) of claim 1 as an active ingredient. 15 7. A method of preventing or treating infectious diseases caused by <? / Os / nzó'a »pez / róígemç characterized by comprising the administration of bacteriophage ΦΟ22 (KCCM11364P) of claim 1 or the composition of. claim 2, for animals, except humans. 8. The method according to claim 7, characterized by the fact that the infectious disease 20 be necrotic enteritis.